Identification and gene disruption of small noncoding RNAs in Streptomyces griseus.

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan.

Abstract

Small noncoding RNAs (sRNAs) have been shown to control diverse cellular processes in prokaryotes. To identify and characterize novel bacterial sRNAs, a gram-positive, soil-inhabiting, filamentous bacterium, Streptomyces griseus, was examined, on the assumption that Streptomyces should express sRNAs as important regulators of morphological and physiological differentiation. By bioinformatics investigation, 54 sRNA candidates, which were encoded on intergenic regions of the S. griseus chromosome and were highly conserved in those of both Streptomyces coelicolor A3(2) and Streptomyces avermitilis, were selected. Of these 54 sRNA candidates, 17 transcripts were detected by Northern blot analysis of the total RNAs isolated from cells grown on solid medium. Then, the direction of transcription of each sRNA candidate gene was determined by S1 nuclease mapping, followed by exclusion of four sRNA candidates that were considered riboswitches of their downstream open reading frames (ORFs). Finally, a further sRNA candidate was excluded because it was cotranscribed with the upstream ORF determined by reverse transcription-PCR. Thus, 12 sRNAs ranging in size from 40 to 300 nucleotides were identified in S. griseus. Seven of them were apparently transcribed in a growth phase-dependent manner. Furthermore, of the 12 sRNAs, the expression profiles of 7 were significantly influenced by a mutation of adpA, which encodes the central transcriptional regulator of the A-factor regulatory cascade involved in both morphological differentiation and secondary metabolism in S. griseus. However, disruption of all 12 sRNA genes showed no detectable phenotypic changes; all the disruptants grew and formed aerial mycelium and spores with the same time course as the wild-type strain on various media and produced streptomycin similarly to the wild-type strain.

Outline of the procedures used in this study. (A) Flowchart of the procedures for identifying sRNA genes. Each step is shown on the left, and the number of sRNA candidates selected in each step is given on the right. (B) Schematic representation of the experimental procedures used in this study. As an example, an IGR between SGR3322 and SGR3323 is shown. Northern blotting was performed to verify transcription from the IGR, followed by S1 nuclease mapping to determine the direction of the transcript and to estimate the transcriptional start point of the sRNA. The monocistronic transcriptional unit in the IGR was confirmed by RT-PCR analysis. To investigate the in vivo function of the sRNA, the sRNA gene was deleted from the chromosome of S. griseus.

Experimental verification of predicted sRNAs by Northern blot analysis. Shown are Northern blots hybridized with 32P-labeled DNA probes. RNA was prepared from cells grown at 28°C on cellophane on the surface of YMPD agar for the times indicated above each blot. All Northern blots were run using the same RNA samples. The RNA samples (30 μg) were separated by denaturing polyacrylamide gel electrophoresis. 5S rRNA, which was stained with ethidium bromide on the gel, was used as an internal control for RNA loading and integrity. For detection of sgs3323, a 0.3-μg RNA sample was used. The amount of sgs3323 was estimated to be more than several hundred times larger than the other sRNAs. Standard markers are shown on the left. Hybridization bands with the predicted sizes are indicated by open circles. Hybridization bands of larger and smaller sizes are indicated by black squares and black triangles, respectively. The analysis was repeated using RNAs prepared from a different culture, and the results were quite reproducible.

RT-PCR experiment to confirm the transcriptional unit of identified sRNAs. RNA prepared from wild-type cells grown at 28°C for 72 h on YMPD agar was used for the RT reaction, using random primers to synthesize a cDNA library. The gene organizations around sRNA genes are classified into three groups (A, B, and C) and schematically represented; the positions and directions of ORFs and sRNAs are represented by black and white arrows, respectively. The regions that we attempted to amplify by PCR are also shown by bars with numerals. The PCR cycling conditions were 98°C for 10 s, 60°C for 30 s, and 72°C for 60 s, and a total of 25 cycles (for sgs2672, sgs2746, sgs3323, sgs3618, sgs4453, sgs4581, sgs5362, sgs5676, sgs6100, and sgs6109) or 30 cycles (sgs3903 and sgs4827) were executed. For amplification of sRNA-specific cDNAs (three in panels A and C and one in panel B), PCR primers were designed within the putative sRNA coding regions, which were predicted by Northern blotting and S1 nuclease mapping. +, with reverse transcriptase; −, without reverse transcriptase (a negative control to show there was no contamination of genomic DNA in the RNA sample).

Predicted secondary structure of sgs3323. The precise transcriptional start and endpoints of sgs3323 were determined by the RACE experiments, showing that the sRNA was 229 nt (Table ). The sgs3323 structure was predicted with the MFOLD program (). A stem-loop structure of a putative transcriptional terminator is present near the 3′-end region.

Time course of sRNA transcription determined by low-resolution S1 nuclease mapping. RNA was prepared from the wild-type (wt) and the ΔadpA mutant strains grown at 28°C on cellophane on the surface of YMPD agar for the times indicated above the blots. As an internal control, the hrdB gene was used. SM, substrate mycelium; AM, aerial mycelium; SP, spore. The ΔadpA mutant grew only as substrate mycelium throughout the growth period. The analysis with RNAs from different cultures was repeated, and the expression profiles of sRNAs were reproducible. Note that the RNA samples used for this S1 nuclease mapping were different from the RNA samples used for the Northern blotting shown in Fig. .